Investigating Small Regulatory RNAs in the S. mutans SloR Regulon

Objectives: Streptococcus mutans is a constituent of the human oral microbiome and the primary causative agent of dental caries, a chronic infectious disease that disproportionately affects populations of lower socioeconomic status. Work in our laboratory is focused on SloR, a 25kDa metalloregulatory protein that modulates S. mutans genes whose products promote cariogenesis. We propose a role for small, regulatory RNAs (sRNAs) as mediators of SloR gene regulation in S. mutans. Based on an in silico analysis of 22 predicted sRNAs, we selected two for further study since they localize to intergenic regions on the UA159 chromosome and harbor putative SloR recognition elements. We noted differential expression of these sRNAs (sRNA14 and sRNA34) in UA159 and a SloR-deficient GMS584 mutant in a S. mutans transcriptome library generated previously in our laboratory.
Methods: Electrophoretic mobility shift assays (EMSAs) were performed to determine whether SloR binds directly to the IGRs containing sRNAs 14 and 34. Expression profiling experiments (qRT-PCR) were also conducted in an effort to validate the S. mutans UA159 and GMS584 transcriptome data. TargetRNA2 was used to identify the predicted downstream targets of sRNAs 14 and 34, the results of which await confirmation in qRT-PCR experiments. A SMARTer smRNA Sequencing Kit is currently being used to generate sRNA sequencing libraries from which additional sRNAs in S. mutans may be identified.
Results: EMSA confirmed a direct interaction between sRNA14 and the SloR protein, and qRT-PCR results support differential sRNA14 and 34 expression in S. mutans UA159 and GMS584, indicating that they are SloR-regulated. The downstream targets of these sRNAs have annotations that are relevant to S. mutans metal ion transport (sloB) and biofilm formation (rgpB).
Conclusions: Select sRNAs in S. mutans are subject to direct SloR control and may play a central role in metal ion homeostasis and biofilm formation, both factors important for cariogenesis.